High temperature lipase, coding gene order and uses thereof

A lipase, high temperature technology, applied in genetic engineering, plant genetic improvement, applications, etc., can solve problems such as poor thermal stability, and achieve the effect of high thermal stability

Inactive Publication Date: 2004-06-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lipase has been isolated from many different microorganisms, such as Penicillium (Huang Jianzhong et al., Industrial Microbiology, 25(3): 1-5, 1995), Geotrichum candidum (Xie Shunzhen, etc., Acta Microbiology, 26(3): 260- 264, 1986), Rhizopus (Jin Qirong et al., Industrial Microbiology, 25(1): 17-20, 1995), Pseudomonas-like alcaligenes (Wu Songgang et al., Acta Microbiology, 37(1): 32-39, 1997 ), Pseudomonas (Kotsuka etc., U.S. Patent 5,846,801, on December 8th, 1998), Candida etc. [William etc., microbial enzyme and biotechnology (Microbial Enzymes and Biotechnology), published by Applied Science SciencePublishers Ltd), 1983, 225-247], most of the lipases have low optimum action temperature (about 35-40°C) and poor thermal stability. When the action substrate is oil with high freezing point, such as hydrogenated oil, beef Oil, tallow tallow, palm oil, etc., such enzymes are not suitable, and lipases with thermostable properties are needed to meet industrial requirements

Method used

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  • High temperature lipase, coding gene order and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Extraction of total DNA from thermophilic anaerobic bacteria MB4

[0022]The thermophilic anaerobic bacterium Thermoanaerobactertengcongensis MB4 isolated from Tengchong Hot Spring, Yunnan, China, was used to get 20 grams of fresh wet thallus, suspend in 10 milliliters of 50mM Tris buffer (pH8.0), add a small amount of lysozyme and 8 milliliters of 0.25mM EDTA ( pH8.0), mix well and place at 37°C for 20min, then add 2 ml of 10% SDS, place at 55°C for 5min, extract with equal volume of phenol and chloroform respectively, take the last supernatant solution, add 2 times Volume ethanol, recover DNA, wash with 70% and absolute ethanol respectively, dissolve the precipitate in 0.5 ml TE buffer solution (pH8.0, 10 mM Tris, 1 mM EDTA), add 10 mg / ml RNase 3 μl, incubate at 37 ° C for 1 hour, use etc. The volume of phenol and chloroform were extracted once, and the supernatant was added with 2 times the volume of ethanol to recover the DNA, washed with 70% and absolute ethanol re...

Embodiment 2

[0028] Purification and Characterization of Recombinant Lipase

[0029] The cells of the recombinant bacteria E.coli DH5αLIP were suspended in 50mM phosphate buffer (pH7), the cells were disrupted by ultrasonic waves, and the centrifuged supernatant was the crude enzyme solution of the recombinant lipase. The supernatant enzyme solution was heated at 70oC for 15 minutes, centrifuged to remove denatured protein, the supernatant enzyme solution was purified by ion exchange column chromatography, hydroxyapatite adsorption column chromatography and PAGE preparative electrophoresis, and the obtained enzyme preparation was prepared in SDS- A band appears on the PAGE. The basic properties of this recombinant lipase were determined using known standard methods of protein chemistry. The molecular weight of the recombinant enzyme measured by SDS-PAGE is 42,000 Daltons, which is similar to the theoretically calculated molecular weight (45,200 Daltons); the reaction temperature of the re...

Embodiment 3

[0031] Hydrolyzed olive oil with recombinant lipase

[0032] Add an appropriate amount of enzyme solution to 50% olive oil solution (prepared with pH7, 0.2M sodium phosphate buffer), heat up to 60° C., and stir at 200 rpm for 16 hours. The oil phase contained fatty acid and the water phase contained glycerol as determined by chromatography, which indicated that the recombinant lipase could hydrolyze olive oil to produce glycerol and fatty acid.

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Abstract

High temperature lipase gene is obtained from Thermoanaerobacter tengcongensis MB4 total DNA to constitute prokaryotic expression plasmid and transferred to colibacillus for expression. Amino acid sequence comparison shows that the lipase is one new kind of lipase.

Description

technical field [0001] The invention belongs to the fields of enzyme genetic engineering and enzyme engineering. Specifically, it relates to a DNA sequence encoding thermophilic anaerobic bacteria (Thermoanaerobacter tengcongensis) MB4 lipase, and also relates to a recombinant plasmid containing the enzyme gene and a recombinant strain expressing the corresponding enzyme. Background technique [0002] Lipase (Lipase, EC3.1.1.3, glyceride hydrolase) is a special hydrolytic enzyme that decomposes fat, catalyzes the decomposition of triglycerides, and produces glycerides, free fatty acids and glycerol. Lipase is water-soluble, and the characteristic of its catalytic reaction is that it can rapidly catalyze and decompose the ester bond at the interface between the insoluble substrate and the water phase, but has little effect on the water-soluble substrate. This point has many advantages in the synthesis of chiral intermediates in organic synthesis. For example, chemical cataly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): F16B37/14B62D25/24
CPCY10T24/309B62D25/24
Inventor 薛燕芬马延和窦岳坦周培瑾
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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